Frusteri Chiacchiera Angelica, Casanova Michela, Bellato Massimo, Piazza Aurora, Migliavacca Roberta, Batt Gregory, Magni Paolo, Pasotti Lorenzo
Department of Electrical, Computer and Biomedical Engineering, University of Pavia, Via Ferrata 5, Pavia, Italy.
Centre for Health Technologies, University of Pavia, Via Ferrata 5, Pavia, Italy.
Sci Rep. 2025 Jan 2;15(1):261. doi: 10.1038/s41598-024-81989-5.
The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.
全球对抗抗菌药物耐药性的竞赛需要新型抗菌药物，这些药物不仅要能有效杀死特定细菌，还要尽量减少新耐药性的出现。最近，有人提出基于CRISPR/Cas的抗菌药物来解决杀伤特异性问题，结果令人鼓舞。然而，由Cas切割引发的靶序列突变的出现被确定为一种逃逸策略，带来了产生新的抗生素耐药基因（ARG）变体的风险。在此，我们评估了一种基于CRISPR干扰（CRISPRi）的抗生素重新致敏策略，该策略可在不损伤靶DNA的情况下抑制基因表达。在重组大肠杆菌中，通过对低拷贝数至高拷贝数的ARG进行单基因和多基因靶向，对包括最后手段药物在内的四种抗生素的耐药性显著降低。逃逸分析证实靶序列中不存在突变，这证实了CRISPRi在新耐药性选择中无有害作用。然后，使用携带具有严重临床关注的ARG的大肠杆菌临床分离株来评估CRISPRi在不同生长条件下的稳健性。美罗培南、黏菌素和头孢噻肟的敏感性在MIC（高达>4倍）和生长延迟（长达11小时）方面以培养基依赖的方式成功提高。ARG抑制在人尿中生长的致病菌株中也有效，这证明了CRISPRi介导的在模拟宿主培养基中的重新致敏作用。本研究为进一步利用CRISPRi作为抗菌剂或研究工具来选择性抑制ARG和研究耐药机制奠定了基础。
